This is a resubmission application for the K99 Pathway to Independence Award. Molecular motor proteins actively transport intracellular cargos in a highly regulated fashion. Cytoplasmic dynein is the largest, most complex, and least understood of the microtubule motor protein families. Because dynein is utilized by the cell for many diverse functions, its activity is highly regulated by a complex web of external protein factors that impinge on the basic mechanochemistry of the motor. As a graduate student, I trained with Dr. Richard Vallee at Columbia University to study the biochemistry and biophysics of cytoplasmic dynein regulation by the neurodevelopmental disease proteins LIS1 and NudE/L. During my postdoctoral career in the lab of Dr. Ronald Vale at the University of California, San Francisco, I have developed new skills in fluorescence single-molecule microscopy. I have used these new skills to discover a novel mode of dynein motility used to slide anti-parallel microtubules apart, a function critical during mitotic spindle assembly. Recently, I have utilized these skills and training to isolate and characterize a stable super-complex of dynein, dynactin, and the adapter protein BicD2 that is over 2MDa in size. I have made the first biophysical measurements of this complex at the single molecule level, revealing unanticipated new motile properties. The goal of this proposal is to understand how dynein regulatory pathways exert proper control of the motor at both the molecular and cellular level. During the mentored K99 phase, this multi-disciplinary proposal aims to: 1) Elucidate the molecular mechanism of dynein motor regulation by divergent regulatory pathways made up of dynactin-BicD2, and Lis1-NudE/L, and 2) Probe the roles of this regulatory activity in the transport of physiological important cargo in a living neuronal system. With the new training and skills acquired in the mentored phase, I will then extend the scope of my research in the independent R00 phase in an effort to understand how dynein processivity regulation is utilized in the coordination of opposite polarity motors, and what effects human neurodegenerative disease mutations have on this coordination. For the experiments proposed in this application, I will acquire additional training in nanometer-precision, multi-color single molecule microscopy and data analysis, Drosophila genetics, DNA nanotechnology, genome engineering, and confocal microscopy in live animals. As co-mentors, Ron Vale and Yuh-Nung Jan will provide expertise in advanced imaging techniques, Drosophila neurobiology and genetics. My collaborators will provide the necessary experience and support in optical trapping microscopy and novel Drosophila genome editing techniques. These new skills and training will afford me the best opportunity to achieve my career goal to launch an independent and successful research laboratory within two years. Overall, the implementation of this proposal will answer long-standing questions in the molecular transport field, and provide novel insight into the mechanism of human neurodegenerative diseases that result from impaired intracellular transport.